Understanding the function of Pga15 family in Candida albicans pathobiology

Objective The largest class of mannoproteins covalently attached to the Candida albicans cell wall are glycosylphospatidylinositol modified-proteins (GPI CWPs) and  66% have unknown functions. As a result of their location at the cell surface, they may be involved in sensing the surrounding environment, maintaining a robust cell wall or interactions with the host. There are 115 predicted GPI CWPs and they include 22 protein families. One family, of uncharacterised proteins, the Pga15 family includes 3 members which are Pga15, Pga41 and Pga42. Moreover, they are only found in C. albicans and C. dubliniensis. Earlier unpublished work by the Munro group has implicated this family in signalling pathways which control the morphological switch from yeast to hyphal form. So, the aim of this project is to define the functional importance of the Pga15 protein family.
Methods hyphal growth induction of C.albicans on poly-L-lysine slides using 10% fetal calf serum (FCS) or N-acetylglucosamine (GlcNAc) and incubated at 37Co for 4 or 6 hours. Furthermore, the fluorescent dye to stain the cell wall e.g Calcofluor-white, Concanavalin A and transmission electro-microscopy (TEM) have been used to have a look at the structure of different cell wall components in different culture media for instance, YPD and NGY. Moreover, biofilm formation into wells in 96-well polystyrene in different media has been conducted and incubated at 37Co for 4, 6 and 24 hours.
Results Mutation of the different family members resulted in alteration in filamentous growth e.g. pga41∆ and pga42∆ had longer hyphae in 10% FCS, while pga42∆ formed shorter hyphae in response to GlcNAc and neutral pH compared to the control strain. Moreover, pga41∆ and pga41∆pga42∆ formed longer hyphae after four hours incubation and pga15∆ had statistically significantly longer hyphal cells at four and six hours compared to control in neutral pH. Furthermore, pga41∆, pga42∆ and pga41∆pga42∆ had a defect in hyperpolarised bud formation in response to hydrogen peroxide. Cell wall specific stains suggested the mutants had significant differences in chitin and mannan levels which were growth medium dependent. High pressure freezing TEM was used to examine the cell wall architecture. The cells were grown in 2 different media YPD and NGY. 
We have found the cell wall architecture differed extensively when cells were grown on different media. For example, NGY-grown cells displayed a shorter outer fibril layer compared to YPD-grown cells, except for the control strain (which had a longer outer layer in NGY). Biofilm formation of the mutants was also assessed and pga42∆ and pga41∆pga42∆ had increased biofilm formation in both RPMI and 10% FCS media compared to the control strain. In addition, pga42∆ are more hydrophobic and  adheres more to laminin compared to the control. 

Conclusion
Absence of Pga15 family members impacts on hyphal growth, biofilm formation and cell wall architecture.

3 / Understanding the function of Pga15 family in Candida albicans pathobiology